1. Field of the Invention
Aspects of the present invention relate to a method and to an instrument for obtaining three-dimensional images of a specimen by microscopy and for obtaining, in three dimensions, the field of fluorescence emitted by this specimen, said specimen being either fluorescent or prelabeled using one or more fluorochromes.
One of the applications of said method and of said instrument relates more particularly to obtaining three-dimensional images of the fluorescence emitted by biological specimens, that may be thick, for the purpose of observing their dynamic behavior and their change over time.
2. Description of the Related Technology
Conventional fluorescence microscopy has provided substantial progress in the field of biology. In particular, the technique of immunofluorescence has made it possible to carry out specific labeling of molecules and their location in tissues. Moreover, the discovery of GFPs (green fluorescent proteins) has revolutionized the study of locating proteins in living cells and of determining the dynamics and interactions of said proteins.
However, one of the problems encountered in conventional fluorescence microscopy is that of the fluorescence emitted by elements lying outside the plane of sharpness. This parasitic light prevents good acquisition of the image by adding substantial background noise. The images become very difficult to interpret when thick biological specimens, such as embryons, are observed.
Confocal microscopy allows this problem to be overcome by exciting the fluorescence of the specimen over a very small area that scans the specimen and by blocking the fluorescence emitted outside the illuminated area by means of a small aperture.
However, confocal microscopy also has its own drawbacks.
One drawback of confocal microscopy is that this technique requires complete scanning of the specimen by an optomechanical device. This operation requires a relatively long time (typically of the order of a few seconds) to analyze a volume before the image is available to be displayed.
Furthermore, the exciting source used in confocal microscopy is generally an argon or argon-krypton laser that may damage biological specimens.
Another drawback is that the exciting sources do not allow the entire wavelength range needed for observation in fluorescence to be easily covered.
Finally, confocal microscopy is an extremely sophisticated technique that requires great precision during manipulation and is also expensive.